Project Summary Mammalian sex chromosomes are enriched with large palindromic sequences harboring gene families expressed almost exclusively in spermatogenic cells, however the molecular functions of these palindromic genes remain largely unknown. Understanding the mechanistic functions of individual palindromic gene families will provide novel insights into genetic factors that contribute to spermatogenic defects (abnormalities in the development of testicular germ cells). The objective of this proposal is to precisely delete individual X- and Y-palindrome gene families to determine their molecular functions during spermatogenesis. This proposal addresses whether deletion of all copy members of an individual X- or Y-palindromic gene family results in spermatogenic defects. This proposition is based on the observation that deletions that remove all members of X- or Y-palindromic gene families result in spermatogenic defects. However, each of these previously studied deletions remove more genes than just an individual palindromic gene family, making it difficult to determine the contribution of individual palindromic gene families to spermatogenic defects. Therefore, we propose to generate precise deletions that remove all members of individual X- or Y-palindromic gene families in mice to understand their role in spermatogenesis. As proof of feasibility, we have generated two, independent, megabase-sized deletions of the Slx and Slxl1 X-palindromic gene families and show that male mice carrying deletions of both gene families are infertile, exhibiting a specific post-meiotic spermatogenic defect (Preliminary Studies). This proposal addresses the importance of X- and Y-palindromic genes via two specific aims: 1) Determine the role of the Slx and Slxl1 X-palindromic gene families in post-meiotic spermatogenesis in male mice carrying deletions of both the Slx and Slxl1 palindrome arrays; 2) Determine the contribution of four additional X- and Y-palindromic gene families in spermatogenesis by individually deleting each of the four distinct gene families. To genetically dissect the functions of individual X- and Y-palindromic gene families in mice, we have developed new chromosome engineering methodologies to efficiently delete large palindromic regions in vivo. Each of the X- and Y-palindromic gene families in mice will be systematically characterized for defects in spermatogenesis. The functions of individual X- and Y-palindromic gene families have been poorly studied in humans and mice because of their recent discovery and complex genomic architecture. The proposed experiments will provide an improved understanding of the roles of individual X- and Y-palindromic genes in male fertility. The fact that X- and Y-palindromes harbor testis-specific gene families makes them ideal candidates for uncovering new genetic factors responsible for human spermatogenic defects. By understanding the molecular functions of X- and Y-palindromic gene families, important insights can be gained into their role in enabling spermatogenesis and how mutations in X- and Y-palindromic gene families disrupt spermatogenesis.